Project Summary/Abstract Cells generate biochemically and morphologically distinct plasma membrane domains by polarizing their internal structure and biosynthetic pathways. Without this polarity, cells could not perform functions such as transport across epithelia and signal transmission in neurons. Further, improper regulation of cell polarity can initiate cancer metastasis, as in unregulated epithelial the mesenchymal transition (EMT). Multiple processes are interwoven for cell polarization, including assembly of a polarized cytoskeleton, synthesis of lipids and proteins with their transport to the appropriate surface, and exocytosis and endocytosis. While long-range transport often involves microtubules, local transport and morphological features of the plasma membrane generally involves an interplay between signaling pathways, microfilaments and membrane traffic. While much is known about each individual area, our two projects address the critical gap in understanding of how they are coordinated. First, we aim to understand how structural elements and signaling pathways converge to define the morphology of a specific membrane domain, using the microvilli on the apical aspect of epithelial cells as our model. We have defined the major structural components and provided insight into regulation of the critical microfilament- membrane linking protein ezrin. We will elucidate the signaling pathways that impinge on ezrin and other factors to restrict microvilli to the apical surface, to determine how microvilli impact the membrane proteome, and to identify the additional functions and regulators of ezrin. Second, we study how motor-based transport along microfilaments is coordinated with membrane traffic. We utilize yeast where microfilaments serve as tracks for the myosin-V based transport of secretory vesicles for bud growth and in organelle segregation between mother and daughter during cell division. We will define fundamental aspects of organelle transport by investigating how the myosin-V picks up secretory vesicles, transports and delivers them in coordination with vesicle biogenesis and exocytosis. We have established a system with high temporal and spatial precision for imaging the delivery cycle of a molecular motor, as well as steps in exocytosis, and shown that motor release is coordinated with, and dependent on, exocytosis. We will undertake an extensive mechanistic analysis of the timing, dependencies, and coordination of steps in exocytosis and motor release exploiting available and newly generated mutants. As the molecules involved are conserved between yeast and vertebrates, most notably identified from the extensive studies of neurotransmitter release at the synapse, the findings will be of general significance. It is important to note that exocytosis in yeast is orders of magnitude slower that at the neuromuscular junction, permitting far greater temporal resolution, and in a much more experimentally accessible system. Moreover, many diseases are associated with defects in molecular motors, including myosin-Vs, and components involved in exocytosis. The proposed research will provide fundamental and medically relevant insights into two critical aspects of cell polarity, local regulation of cell morphology and organelle delivery.